Modulation of ionizing radiation-induced apoptosis by bantam microRNA in Drosophila

In Drosophila, heterozygosity in the pro-apoptotic gene hid significantly reduces apoptosis that is induced by ionizing radiation (IR). Therefore, mechanisms that regulate Hid levels can potentially contribute to life-or-death decision of an irradiated cell. 3′UTR of hid mRNA contains 5 potential binding sites for bantam microRNA. Ectopic expression of ban attenuated apoptosis that results from ectopic expression of hid but the significance of this regulation under physiological conditions remained to be investigated. We report here that ban is needed to limit IR-induced apoptosis in larval imaginal discs. Using tubulin–EGFP ban sensors with ban consensus sequences in the 3′UTR, we find that EGFP decreases following IR, indicating that IR activates ban. Likewise, a tubulin–EGFP reporter with hid-3′UTR is repressed in irradiated discs and this repression requires ban consensus sites in the hid 3′UTR. ban mutant larvae show increased sensitivity to killing by IR, which is suppressed by a mutation in hid. These results can fit into a model in which IR activates ban and ban represses hid to limit IR-induced apoptosis. miRNAs have been shown previously to be induced by radiation but this is the first report that a miRNA is functionally important for radiation responses.     

Analysis of the shortvein cis-regulatory region of the decapentaplegic gene of Drosophila melanogaster

In mammals, the Transforming Growth Factor-beta (TGF-beta) superfamily controls a variety of developmental processes. In Drosophila, by contrast, a single member of the superfamily, decapentaplegic (dpp) performs most TGF-beta developmental functions. The complexity of dpp functions is reflected in the complex cis-regulatory sequences that flank the gene. Dpp is divided into three regions: Hin, including the protein-coding exons; disk, including 3' cis-regulatory sequences; and shortvein (shv), including noncoding exons and 5' cis-regulatory sequences. We analyzed the cis-regulatory structure of the shortvein region using a nested series of rearrangement breakpoints and rescue constructs. We delimit the molecular regions responsible for three mutant phenotypes: larval lethality, wing venation defects, and head capsule defects. Multiple overlapping elements are responsible for larval lethality and wing venation defects. However, the area regulating head capsule formation is distinct, and resides 5' to these elements. We have demonstrated this by isolating and describing two novel dpp alleles, which affect only the adult head capsule.     

Peptidoglycan recognition protein LF: a negative regulator of Drosophila immunity

Peptidoglycan recognition proteins (PGRPs) play important roles in the innate immune defence. Each PGRP detects a distinct subset of peptidoglycans and initiate immune signalling or enzymatic degradation of peptidoglycans. Here we characterize one of the 13 Drosophila PGRPs, PGRP-LF. PGRP-LF is membrane bound and has its two PGRP domains, z and w, localized outside the cell. Our data demonstrate that the z-and w-domain differ in their affinities to peptidoglycan. The z-domain has affinity to several groups of peptidoglycans while the w-domain only recognizes peptidoglycan from Escherichia coli. In addition, we observed that overexpression of PGRP-LF in Drosophila melanogaster Schneider 2 cells (S2 cells) promotes aggregation of cells. Furthermore, following immune stimulation of S2 cells overexpressing PGRP-LF, we noticed a reduced up-regulation of expression of antimicrobial peptide genes, in consonance with an immune suppressive role for PGRP-LF.     

Genetic mosaic analysis of decapentaplegic and wingless gene function in the Drosophila leg

 Genetically mosaic flies were constructed which lack a functional decapentaplegic (dpp) or wingless (wg) gene in portions of their leg epidermis, and the leg cuticle was examined for defects. Although dpp has previously been shown to be transcribed both ventrally and dorsally, virtually the only dpp-null clones that affect leg anatomy are those which reside dorsally. Conversely, wg-null clones only cause leg defects when they reside ventrally – a result that was expected, given that wg is only expressed ventrally. Both findings are consistent with models of leg development in which the future tip of the leg is specified by an interaction between dpp and wg at the center of the leg disc. Null clones can cause mirror-image cuticular duplications confined to individual leg segments. Double-ventral, mirror-image patterns are observed with dpp-null clones, and double-dorsal patterns with wg-null clones. Clones that are doubly mutant (null for both dpp and wg) manifest reduced frequencies for both types of duplications. Duplications can include cells from surrounding non-mutant territory. Such nonautonomy implies that both dpp and wg are involved in positional signaling, not merely in the maintenance of cellular identities. However, neither gene product appears to function as a morphogen for the entire leg disc, since the effects of each gene’s null clones are restricted to a discrete part of the circumference. Interestingly, the circumferential domains where dpp and wg are needed are complementary to one another. Received: 25 March 1996 / Accepted: 13 June 1996     

D-cbl, a negative regulator of the Egfr pathway, is required for dorsoventral patterning in Drosophila oogenesis

During Drosophila oogenesis, asymmetrically localized Gurken activates the EGF receptor (Egfr) and determines dorsal follicle cell fates. Using a mosaic follicle cell system we have identified a mutation in the D-cbl gene which causes hyperactivation of the Egfr pathway. Cbl proteins are known to downregulate activated receptors. We find that the abnormal Egfr activation is ligand dependent. Our results show that the precise regulation of Egfr activity necessary to establish different follicle cell fates requires two levels of control. The localized ligand Gurken activates Egfr to different levels in different follicle cells. In addition, Egfr activity has to be repressed through the activity of D-cbl to ensure the absence of signaling in the ventral most follicle cells.     

Drosophila Src42A is a negative regulator of RTK signaling

The Src family of nonreceptor tyrosine kinases has been implicated in many signal transduction pathways. However, due to a possible functional redundancy in vertebrates, there is no genetic loss-of-function evidence that any individual Src family member has a crucial role for receptor tyrosine kinase (RTK) signaling. Here we show that an extragenic suppressor of Raf, Su(Raf)1, encodes a Drosophila Src family gene Src42A. Characterization of Src42A mutations shows that Src42A acts independent of Ras1 and that it is, unexpectedly, a negative regulator of RTK signaling. Our study provides the first evidence that Src42A defines a negative regulatory pathway parallel to Ras1 in the RTK signaling cascade. A possible model for Src42A function is discussed.     

Robustness of the Dpp morphogen activity gradient depends on negative feedback regulation by the inhibitory Smad, Dad

Developmental patterning relies on morphogen concentration gradients, which generally provide invariable positional information despite genetic fluctuations. Theoretical studies have predicted robust patterning; however, little experimental evidence exists to support this idea. In this report, we examine the robustness of the Decapentaplegic (Dpp) (a Drosophila homologue of bone morphogenetic protein [BMP]) activity gradient in the presence of fluctuations in Dpp receptor levels. Dpp activity can be measured by the degree of phosphorylation of Mothers against dpp (Mad), a major signal transducer. We determined that phosphorylated Mad (pMad) levels remain constant when an extra copy of thickveins (tkv), which encodes the receptor, is introduced into the wild-type background. Higher Tkv levels, expressed under the control of an artificial promoter, result in constant pMad levels. This prompted us to study the mechanisms that underlie pMad level maintenance even when Tkv levels are increased. We focused on the inhibitory Smad, daughters against dpp (dad), which is induced by Dpp signaling and negatively regulates Dpp activity. In the absence of dad, pMad levels significantly increase when Tkv levels increase. These results suggest that Dpp activity gradient robustness when Tkv levels increase depends, at least in part, on negative feedback regulation by dad.     

The Drosophila fragile X protein functions as a negative regulator in the orb autoregulatory pathway

Translational regulation of maternal mRNAs in distinct temporal and spatial patterns underlies many key decisions in developing eggs and embryos. In Drosophila, Orb is responsible for mediating the translational activation of mRNAs localized within the developing oocyte. Orb is a germline-specific RNA binding protein and is one of the founding members of the CPEB family of translational regulators. Here we show that Orb associates with the Drosophila Fragile X Mental Retardation (dFMR1) protein as part of a ribonucleoprotein complex that controls the localized translation of mRNAs in developing egg chambers. One of the key orb regulatory targets is orb mRNA, and this autoregulatory activity is critical for ensuring that Orb protein is expressed at high levels in the oocyte. We show that dFMR1 functions as a negative regulator in the orb autoregulatory circuit, downregulating orb mRNA translation.     

Yorkie drives supercompetition by non-autonomous induction of autophagy via bantam microRNA in Drosophila

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Expression analysis of decapentaplegic gene in the silkworm,Bombyx mori

The decapentaplegic (dpp) gene in Drosophila is involved in multiple developmental processes, and is a highly conserved among various eukaryotic species, including Bombyx mori. Although the gene has well been characterized in Drosophila species the B. mori dpp has not yet been functionally analyzed. In this study, we analyzed the expression pattern of B. mori dpp in 12 different developmental days/stages (7 days for fifth instar larvae, 2 days for spinning stage, 2 days for pupal stages, and 1 day for adults) in both male and female silkworms using quantitative real‐time RT‐PCR (qRT‐PCR). mRNA expression of B. mori dpp was much higher in the female larvae up to the mid‐stage of the fifth instar compared with the corresponding male larvae. Similarly, dpp expression also was much higher in females during the eclosion period than that in the corresponding male pupae. During the embryonic stage, the expression level of the dpp gene was much higher compared to that of adult stage in both male and female silkworms. These results suggest that the B. mori dpp gene plays multiple roles in the developmental of B. mori.     

Negative regulation of diminutive cancer regulator through differentiation and microRNA pathway components in Drosophila cells

Drosophila model is intensively studied for the development of cancer. The diminutive (dMyc), a homolog of the human MYC gene, is responsible for cell- apoptosis and its upregulation is responsible for determining the fate of cancerous growth in humans and Drosophila model. This work implores the requirement of dMyc and its expression as one of the major regulator of cancer with other proteins and repression of dMyc mRNA in Drosophila S2 cells. Here we report protein complex of Argonaute 1 (AGO1), Bag of marbles (Bam), and Brain tumor (Brat) proteins and not the individual factor of this complex repression of dMyc mRNA in Drosophila Schneider 2 cells and promote differentiation in cystoblast of Drosophila ovary. These results exhibit the significant role of this complex, including master differentiation factor Bam with other various differentiation factor Brat and microRNA pathway component AGO1, which may negatively regulate dMyc mRNA and so the dMyc protein.     

Endogenous transcripts direct microRNA degradation in Drosophila,and this targeted degradation is required for proper embryonic development

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ADI1, a methionine salvage pathway enzyme,is required for Drosophila fecundity

Background

Methionine, an essential amino acid, is required for protein synthesis and normal cell metabolism. The transmethylation pathway and methionine salvage pathway (MTA cycle) are two major pathways regulating methionine metabolism. Recently, methionine has been reported to play a key role in Drosophila fecundity.

Results

Here, we revealed that the MTA cycle plays a crucial role in Drosophila fecundity using the mutant of aci-reductone dioxygenase 1 (DADI1), an enzyme in the MTA cycle. In dietary restriction condition, the egg production of adi1 mutant flies was reduced compared to that of control flies. This fecundity defect in mutant flies was rescued by reintroduction of Dadi1 gene. Moreover, a functional homolog of human ADI1 also recovered the reproduction defect, in which the enzymatic activity of human ADI1 is required for normal fecundity. Importantly, methionine supply rescued the fecundity defect in Dadi1 mutant flies. The detailed analysis of Dadi1 mutant ovaries revealed a dramatic change in the levels of methionine metabolism. In addition, we found that three compounds namely, methionine, SAM and Methionine sulfoxide, respectively, may be required for normal fecundity.

Conclusions

In summary, these results suggest that ADI1, an MTA cycle enzyme, affects fly fecundity through the regulation of methionine metabolism.     

Drosophila Fip200 is an essential regulator of autophagy that attenuates both growth and aging

Autophagy-related 1 (Atg1)/Unc-51-like protein kinases (ULKs) are evolutionarily conserved proteins that play critical physiological roles in controlling autophagy, cell growth and neurodevelopment. RB1-inducible coiled-coil 1 (RB1CC1), also known as PTK2/FAK family-interacting protein of 200 kDa (FIP200) is a recently discovered binding partner of ULK1. Here we isolated the Drosophila RB1CC1/FIP200 homolog (Fip200/CG1347) and showed that it mediates Atg1-induced autophagy as a genetically downstream component in diverse physiological contexts. Fip200 loss-of-function mutants experienced severe mobility loss associated with neuronal autophagy defects and neurodegeneration. The Fip200 mutants were also devoid of both developmental and starvation-induced autophagy in salivary gland and fat body, while having no defects in axonal transport and projection in developing neurons. Interestingly, moderate downregulation of Fip200 accelerated both developmental growth and aging, accompanied by target of rapamycin (Tor) signaling upregulation. These results suggest that Fip200 is a critical downstream component of Atg1 and specifically mediates Atg1’s autophagy-, aging- and growth-regulating functions.     

bantam encodes a developmentally regulated microRNA that controls cell proliferation and regulates the proapoptotic gene hid in Drosophila

Cell proliferation, cell death, and pattern formation are coordinated in animal development. Although many proteins that control cell proliferation and apoptosis have been identified, the means by which these effectors are linked to the patterning machinery remain poorly understood. Here, we report that the bantam gene of Drosophila encodes a 21 nucleotide microRNA that promotes tissue growth. bantam expression is temporally and spatially regulated in response to patterning cues. bantam microRNA simultaneously stimulates cell proliferation and prevents apoptosis. We identify the pro-apoptotic gene hid as a target for regulation by bantam miRNA, providing an explanation for bantam's anti-apoptotic activity.